Background: Microbial agents in root canal systems can induce periodontal inflammation. The aims of this study are to detect anaerobic microorganisms in endodontic-periodontal lesions, determine the genetic diversity among them, and assess the simultaneous colonization of the pulp and periodontal nnicroenvironments by a single clone. Methods: Twenty-seven teeth of patients with endodontic-periodontal lesions were selected. Samples were spread on an agar-blood medium, the detection of each species was performed using a polymerase chain reaction, and the determination of the simultaneous presence of the same species in the microenvironments by one or more clones was determined using arbitrarily primed PCR. Results: Prevotella intermedia (Pi) was the most prevalent species of the colonies in periodontal pockets, whereas Porphyromonas gingivalis (Pg) and Pi were the more prevalent in root canals. Isolates of Pi and Pg were simultaneously identified in root canals and periodontal pockets. Eighteen percent of teeth exhibited the simultaneous colonization by Pg, Tannerella forsythia (previously T. forsythensis), and Porphyromonas endodontalis in the pulp and periodontal nnicroenvironments. The presence of these species was noted even in niches from which no colonies were isolated. Seventeen different genotypes were found in periodontal and pulp sites, with the majority of sites colonized by one or two different genotypes. A high degree of genotype similarity was found for samples of Pg isolated from only one site as well as for those isolated from both microenvironments. Conclusion: Different clones of Pi and Pg with a high intraspecific genotype similarity were found to colonize the same anatomic sites in endodontic-periodontal infections. J Periodontol 2011;82:1767-1775.821217671775Brazilian fostering agency Research Support Foundation of the State of Sao Paulo, Sao Paulo, SP, Brazil [2003/10423-4]National Council for Scientific and Technological Development (Brasilia, Federal District, Brazil)Brazilian fostering agency Research Support Foundation of the State of Sao Paulo, Sao Paulo, SP, Brazil [2003/10423-4]